Native hirudin constitutes a group of nearly identical polypeptides with a molecular weight around 7000 Daltons and consisting of 65 to 66 amino acids that are produced in the salivary glands of the leech Hirudo medicinalis [Markwardt F (1970) Methods in Enzymology; 19: 924-932]. Hirudin is the most potent natural inhibitor of the serine protease thrombin, with a Ki of 10−11 to 10−14 M. It forms a 1:1 complex at the active site of thrombin resulting in inhibition of its proteolytic activity [Johnson P H, et al (1989) Sem. Thromb. Hemost.; 15: 320-315; Stone S R, & Hofsteenge J. (1986) Biochemistry; 4622-46242,3]. The X-ray crystal structure of hirudin and the hirudin/thrombin complex have been solved. The globular N-terminal domain of hirudin binds to the active-site cleft of thrombin, while the C-terminal tail binds to the fibrinogen binding exosite of thrombin. Site directed mutagenesis studies have been carried out with both hirudin and the hirudin/thrombin complex [Grütter M G et al (1990) EMBO Journal; 9: 2361-2365; Rydel T J, et al (1990) Science; 249:277-280; Rydel T J, et al (1991) Journal of Molecular Biology; 221: 583-601].
Recombinant hirudins (r-hirudin) differ from naturally occurring hirudin by the lack of a sulphate group at tyrosine 63 and the first two N-terminal amino acids [Märki W E, et al (1997) Semin. Thromb. Haemost; 17: 88-93]. Preclinical and clinical studies have shown that r-hirudin is an effective anticoagulant for prevention and treatment of venous thromboembolism in patients undergoing elective hip surgery and for patients with chronic stable coronary heart disease, acute myocardial infarction, unstable angina pectoris as an adjunct to coronary angioplasty, and in combination with intracoronary thrombolysis [Lieber V, et al (2002) Seminars in Thrombosis and Hemostasis; 28: 483-489]. Recent studies have also suggested that hirudin could be used in stroke therapy [Karabiyikoglu M, et al (2004) Journal of Cerebral Blood Flow & Metabolism. 24: 159-166].
The therapeutic potential of hirudin based thrombin inhibitors has been demonstrated by the development of Desirudin [Iprivask, Aventis Inc.]; [Val1, Val2]-63-desulphohirudin and Lepirudin [Refludan, Schering/Berlex]; [Leu1, Thr2]-63-desulphohirudin.
Desirudin was first approved in the EU for thrombosis prophylaxis after orthopaedic hip and knee surgery. In April 2003 it was approved by the FDA and is now marketed as Iprivask. Lepirudin is approved in both the European Union (1997) and the United States (1998) for the treatment of patients suffering from heparin-induced thrombocytopaenia (HIT) [Greinacher A, et al (1999). Circulation; 99: 73-80; Greinacher A, Janssens U, Berg G et al. (1999) Circulation; 99: 587-593; Harenberg J, et al (1997) Seminars in Thrombosis and Hemostasis; 23: 189-196]. Heparin is currently used to prevent blood clots in more than 20 million patients per year in Europe and the US. In the US and EU alone, approximately 500,000 patients per year are potential candidates for lepirudin therapy.
Despite its evident clinical utility, a significant problem associated with the clinical use of hirudin is its immunogenicity. Anti-hirudin antibodies can be detected in patients who have received lepirudin and desirudin [Song X, et al (1999) Circulation; 100: 1528-1532; Eichler P, et al (2000) Blood; 96: 2373-2378; Greinacher A, et al (2003) Blood; 101: 2617-2619; Kischer K-G, et al (2003) Thromb. Haemost; 89: 973-982; Eichler P, et al (2004) Blood; 103: 613-616]. Moreover, lepirudin can cause fatal anaphylaxis, particularly in HIT patients who are treated within 3 months of a previous exposure. Between 1994 and 2002, 9 patients have been judged to have had severe anaphylaxis in close temporal contact with lepirudin. All reactions occurred within minutes of intravenous lepirudin administration, with 4 fatal outcomes. In all 4 cases, a previous uneventful treatment course with lepirudin was identified (1 to 12 weeks earlier). High-titre IgG anti-lepirudin antibodies were recorded in an additional patient with anaphylaxis [Greinacher A, Lubenow N, Eichler P (2003). Circulation; 108: 2062-2065].
Generation of a high affinity long lived antibody response, and in particular a response that can be seen to have undergone class switching to high titre IgG antibody, is dependent on CD4+ T-cell help. T cell receptor (TCR) binding of peptide epitopes presented in the context of MHC Class II on the surface of antigen presenting cells (APCs), will lead to T-cell proliferation and the production of cytokines that can modulate immune responses. Patients who develop antibodies to hirudin□ have T-cells that are capable of recognising peptide fragments of hirudin bound to MHC class II molecules.
The present invention is concerned with the identification of T-cell epitopes in hirudin and with hirudin molecules in which amino acid substitution and or combinations of substitution have been conducted. The substitutions confer a reduced immunogenic profile on the protein whilst retaining functional activity.